A recent genome-wide association study (GWAS) found that single nucleotide polymorphisms (SNPs) in CR1 exhibited the third highest association with genetic risk for Alzheimer's disease (AD), following only ApoE and ApoJ. Three non-overlapping GWAS efforts have confirmed a highly significant CR1 SNP association with AD risk. The overall goal of the present project is to fill in two major gaps left by the GWAS reports. 1) How is CR1 functionally altered in AD? and 2) To what extent do CR1 polymorphisms account for the functional alterations? These two issues are linked in our proposal by a reverse-engineering strategy that will first assess functional CR1 changes in AD, then use that information to identify the responsible CR1 SNPs. Specific Aim 1. Identify abnormalities in CR1 protein molecular weight, expression, binding, or function in AD and ND subjects. Despite its association with AD risk, CR1 is actually very poorly expressed in brain. Instead, its primary roles are regulation of spontaneous complement activation and clearance of pathogens and immune complexes (ICs)-both of which occur almost exclusively in the peripheral circulation. Aim 1 will therefore evaluate CR1 structure, expression, binding, complement regulation, and pathogen/IC clearance in blood samples from 300 AD and 300 ND subjects using ELISA, Western blot, complement activation and other techniques with which the investigators have documented expertise. SNPs themselves do not cause disease risk;functional changes induced by SNPs do. Testing mechanisms of CR1 function in AD is therefore an essential first step in understanding the full significance of AD-related CR1 polymorphisms. Specific Aim 2. Test the extent to which the CR1 protein abnormality or abnormalities identified in Aim 1 are linked to specific CR1 gene polymorphisms. Previous GWAS analyses were able to sample only a small fraction of the 476 known SNPs in CR1, and the CR1 SNPs they identified turn out to be in non- coding regions. The elucidation of exact, risk-enhancing SNPs, however, entails many well known challenges and thousands of samples. We have therefore chosen the more modest goal of seeking functional correlates. Using the Aim 1 samples, a RainDance enrichment approach will be employed to select for the CR1 genomic region, followed by bar-coded next generation sequencing on the Illumina GA HiSeq instrument. CR1 SNPs will then be tested, within subjects, for association with any CR1 protein alterations found in Aim 1. A potential shortcut lies in the fact that nearly a dozen CR1 SNPs have already been linked to specific CR1 protein abnormalities in other disease contexts. Candidate SNPs will be further evaluated by site-directed mutagenesis to determine which of them recapitulates Aim 1 CR1 protein defects. An ultimate goal will be to perform validation and true risk assessment in a much larger sample population, an undertaking that would be warranted and facilitated by our prior identification of CR1 SNPs that induce specific CR1 functional alterations. PUBLIC HEALTH RELEVANCE: Large-scale studies have shown that possession of variants of the complement receptor 1 (CR1) gene is one of the greatest genetic risk factors for Alzheimer's disease. However, it is uncertain which CR1 gene variants are responsible, and little is known about how CR1, which is poorly expressed in the brain, might play a role. The proposed research seeks to identify specific functional abnormalities of CR1 in Alzheimer's disease, then link those abnormalities back to the gene variants that may cause them. By doing so, the true significance of CR1 to Alzheimer's can be understood, and treatments to address CR1 defects can be rationally developed.